The endoplasmic reticulum (ER) is the compartment where membrane and secretory proteins are modified, folded, and assembled. Part of the folding process for most extracellular proteins includes formation of disulfide bonds which can add stability to folded polypeptides and can link subunits of protein complexes. The formation of disulfide bonds requires both enzymes for the generation of disulfide bonds in the lumen of the ER as well as a pathway for the transfer of these bonds to substrate proteins. Dr. Kaiser's research group proposes a combination of biochemical, structural, and genetic experiments in the yeast S. cerevisiae to give fundamental insight into the mechanisms of disulfide bond formation in living cells. Previous work in Dr. Kaiser's laboratory has delineated the core pathway in the ER for protein disulfide bond formation in which a luminal oxidase Ero1p (or Erv2p) transfers a disulfide bond to protein disulfide isomerase (PDI) which in turn transfers its disulfide bond to a substrate protein. Structural studies of Ero1p and Erv2p reveal that although these proteins are not similar in sequence, they nevertheless share key structural features giving insight into the mechanisms by which disulfide bonds are generated and transferred from one protein to another. In this application, Dr. Kaiser proposes to determine how disulfide bonds are selectively transferred from Ero1p to PDI. Additional experiments are designed to understand how the functions of ER oxidases are integrated into the redox biochemistry of the cell. Studies of disulfide bond formation in yeast will be extended to understand interesting disulfide bond forming processes in other organisms including G. lamblia and C. elegans. In S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to uncover the genes and proteins responsible protein folding in the ER. A detailed understanding of these pathways in S. cerevisiae will make it possible to understand parallel processes in mammalian cells, opening the way to diagnose dysfunctional folding in the ER of mammalian cells and providing new opportunities to control the assembly and secret on of extracellular proteins.